Synaptic Mutant Huntingtin Causes Synaptic Dysfuntion Through Inhibiting Synapsin-1Phosphorylation

Huntington’s disease (HD) is an inherited neurodegenerative disease. Clinically, Huntington’s disease is characterized by irrepressible motor dysfunction, psychiatric disturbances and cognitive deterioration to dementia. The pathological characteristic feature of HD is selectively neurodegeneration preferentially in striatal and cortex neurons. HD is caused by CAG repeats (>35CAGs) in exon1of huntingtin gene which generates mutant huntingtin (mHtt) with a polyglutamine (polyQ) expansion. Normal huntingtin mainly expresses in cytoplasm of neuron, and is associated with membrane structure and organelle, such as mitochondrion, Golgi, endoplasmic reticulum, endosome and autophagic vacuoles. Mutant huntingtin leads to formation of aggregates in cytoplasma and nucleus and the disease progresses over time. We know that mutant htt in different subcellular regions affects multiple cellular functions. For example, mutant htt in the nucleus can affect gene expression, mitochondrial function, intracellular trafficking, and metabolism. Considering neuronal cells have unique, long neuronal processes and synapses that may be vulnerable to toxic proteins, many studies have focused on synaptic function, revealing that synaptic dysfunction is the common pathological event in a variety of neurodegenerative diseases. Although synaptic dysfunction in HD mouse models has been well-documented, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remains to be determined. Since synapses are unique to neuronal cells, understanding whether synaptic mHtt could cause age-dependent progression of neurological symptoms and early death, or synaptic mHtt caused neuropathology and inhibited neurotransmitter release is critical for unraveling the selective neuropathology seen in HD.Therefore, we fused exon1htt containing normal (20Q) or150Q (150Q) to synaptosomal-associated protein25(SNAP25) to generate SNAP25-htt transgenic mice:SNAP25-Htt150Q and SNAP25-Htt20Q as control. And we studied these transgenic mice which selectively express huntingtin in the presynaptic terminals, to understanding whether and how synaptic mutant huntingtin directly mediates HD neuropathology.Transgenic mutant htt selectively accumulated in the presynaptic terminals in transgenic mice. We used SNAP25-Htt20Q/150Q for pronuclear injection of mouse embryos. Then we selected the SNAP25-Htt20Q/150Q lines that could be bred to F1generation and showed the clear expression of transgene. Genomic DNA and Western Blot analysis revealed that transgenic mutant htt was expressed at a lower level than control SNAP25-Htt20Q and was not overexpressed compared to endogenous mutant htt.To better assess the relative levels of transgenic htt in presynaptic terminals, we performed subcellular fractionation, which could enrich presynaptic proteins, such as SNAP25. The results showed that both SNAP25-Htt20Q and SNAP25-Htt150Q were enriched in the presynaptic fraction. Importantly, degraded htt fragments were also present in the presynaptic fraction.We performed immunofluorescent staining, which could better reveal the subcellular localization of proteins. SNAP25-Htt150Q was clearly present as puncta outside the nuclei of neuronal cells in the cortex and striatum. To verify that mutant htt was indeed localized in the presynaptic terminals, we performed electron microscopy (EM). EM examination revealed the presence of htt aggregates in the axons and presynaptic, but not postsynaptic terminals. Since Western Blot had demonstrated that these htt fragments were not labeled by anti-SNAP25, we further verified that cleaved htt fragments were responsible for forming synaptic aggregates, the majority of synaptic aggregates were not labeled by anti-SNAP25. In the mouse brain and in the synapses of transfected neurons, the negative staining of the majority of htt aggregates suggested that the most htt aggregates in the mouse brain were formed by cleaved htt fragments lacking SNAP25. Also, these aggregates were not associated with the presynaptic plasma membrane, perhaps because they were formed by small huntingtin fragments without SNAP25.Thus, using fractionation assays, immunofluorescent staining and EM, we found convincing evidence that transgenic htt was selectively accumulated in the presynaptic terminals in transgenic mice and this accumulation was age-dependent and largely mediated by cleaved htt fragments without SNAP25. Therefore, we generated novel SNAP25-Htt20Q/150Q transgenic mouse model that selectively express mutant huntingtin in synapses.Age-dependent progression of neurological symptoms in SNAP25-Htt150Q transgenic mice. In order to value whether transgenic SNAP25-Htt150Q mice showed progressive neurological phenotypes similar to other HD transgenic mice, we observed the SNAP25-Htt150Q transgenic mice to find some phenotype such as reduced body weight, clasping, and hunchback appearance. These symptoms occurred in old mice in an age-dependent manner and importantly, did not occur in SNAP25-Htt20Q mice that expressed a higher level of SNAP25-Htt20Q than SNAP25-Htt150Q, indicating that polyQ expansion, rather than tagging htt with SNAP25, was responsible for the neurological symptoms. Strikingly, transgenic SNAP25-Htt150Q mice die earlier than WT and SNAP25-Htt20Q transgenic mice.Using rotarod performance assessment, acoustic startle responses, the light-dark box test and the beam-walking assay to test the transgenic mice phenotype, our findings showed that SNAP25-Htt150Q mice recapitulated the neurological symptoms as other established HD transgenic mice.Synaptic mutant huntingtin caused neuropathology and inhibited neurotransmitter release. It remained unclear whether synaptic mutant htt could cause early neuropathology, such as reactive gliosis which was an early sign of neurodegeneration due to glial proliferation in response to neuronal injury. We therefore stained the brains of our HD mouse models using an antibody against glial fibrillary acidic protein (GFAP), an astrocytic marker protein. We found that there was an obvious increase in GFAP staining in SNAP25-Htt150Q transgenic mouse brain regions, including the cortex, striatum, corpus callosum, and cerebellum. Western Blot and quantitation of the ratio of GFAP to GAPDH also verified the increased astrocytic gliosis in the SNAP25-Htt150Q mouse striatum versus WT and SNAP25-Htt20Q transgenic mouse brains. These results suggested that mutant htt in presynaptic terminals could mediate neuronal injury and neuropathological changes.Electrophysiological studies of corticostriatal slices from mice had supplied an approach to test glutamate release. Given the presynaptic localization of transgenic mutant htt in SNAP25-Htt150Q mice, it was important to examine whether mutant htt affected synaptic neurotransmitter release. Evaluation the strength of synaptic transmission by measuring the field excitatory post-synaptic potentials (fEPSPs) in the sensorimotor cortex of the mouse brain slices, the amplitude of paired-pulse facilitation (PPF), and examination of the long-term potentiation (LTP) of fEPSPs indicated that the expanded glutamine repeat in SNAP25-Htt150Q had specific toxicity, and the pre-synaptic function, especially the capacity for glutamate release, which was impaired in SNAP25-Htt150Q mice.To verify the reduction in glutamate release occurred in the striatum, we isolated the mouse brain striatal slices and performed glutamate release assays using [3H]glutamate. We found that glutamate release was selectively reduced in the SNAP25-Htt150Q slices, and [3H]GABA release did not appear to be significantly impacted, suggesting that vesicular release of glutamate was more vulnerable to synaptic mutant htt.PC12cells provided a cellular model to measure vesicular dopamine release that was dependent on synapsin-1phosphorylation. We measured [3H]dopamine release from these PC12cells and found that mutant htt indeed inhibited dopamine release.Mutant htt bound to C-terminal synapsin-1. Because it was selectively localized in the presynaptic terminals, transgenic mutant htt may abnormally bound synaptic vesicles to impair synaptic transmission. To test this hypothesis, we used synaptosomes isolated from WT mouse cortex, and then incubated them with the lysates of transfected PC12cells, which stably expressed either normal (20Q) or polyQ expanded (150Q) exon1htt. We found that more mutant htt (150Q) than normal htt (20Q) bound synaptosomes. To explore how mutant htt bound more synaptosomes, we wanted to use htt immunoprecipitates and mass spectrometry to identify synaptic proteins that bound mutant htt. We isolated synaptosomes from SNAP25-Htt150Q mouse brains, and then immunoprecipitated synaptic proteins with an antibody to htt (mEM48). The precipitated proteins were subjected to mass spectrometry, which identified578proteins and1732peptides. This experiment revealed that synapsin-1, a presynaptic vesicle protein that plays an important role in neurotransmitter release from vesicles was a good candidate to associate with mutant htt, as it was increased in SNAP25-Htt150Q immunoprecipitates. To verify that mutant htt associates with synapsin-1in vivo, we performed htt immunoprecipitation using brain tissues from our newly generated HD mouse, HD CAG150Kl mouse and PC12stable cell lines. All of these htt immunoprecipitation supported the idea that mutant htt in synapse bound more tightly to synapsin-1.Synapsin-1consists of two isoforms,1a and1b, which contains a number of phosphorylation sites and a proline-rich domain at their C-terminal region. Because the proline-rich domain can serve as a binding region for vesicle trafficking, we generated a truncated synapsin-1(tsynapsin) by deleting the C-terminal proline-rich region and expressed it with exon1-20Q or exon1-150Q htt in transfected HEK293 cells. Immunoprecipitation of htt revealed that, while both synapsin-1a and-1b could associate with htt, depletion of the proline domain eliminated the association of synapsin-1with htt.Synaptic mutant htt inhibited synapsin-1phosphorylation. Phosphorylation of synapsin-1critically regulates the function of synapsin-1, and S9phosphorylation in the N-terminal region of synapsin-1is important for synaptic vesicle neurotransmitter release. By expressing truncated synapsin-1lacking this C-terminal region in HEK293cells, we then tested if mutant htt bound the C-terminal region of synapsin-1to affect synapsin-1phosphorylation. Comparing the phosphorylation of N-terminal synapsin-1at serine9(S9) of full-length synapsin-1b and truncated synapsin-1(tsynapsin), we found that while S9phosphorylation in full-length synapsin-1was inhibited by htt-150Q, the same phosphorylation in tsynapsin was not affected by htt-150Q as compared to htt-20Q. Thus, the inhibition of synapsin-1phosphorylation by mutant htt depended on the interaction of mutant htt with C-terminal region of synapsin-1.To investigate whether mutant htt could affect the phosphorylation of endogenous synapsin-1, we performed Western Blot analysis of PC12cells and HD mouse brains with antibodies to different phosphorylated synapsin-1, as the abnormal interaction of mutant htt with synapsin-1could also affect other phosphorylation sites of synapsin-1. We compared endogenous synapsin-1phosphorylation in PC12cells expressing exon-1150Q htt via Western Blot and specific antibodies to different phosphorylation sites in synapsin-1and found a reduction in S9, S549, and S603phosphorylation in synapsin-1. It was important to validate this finding in the HD mouse brain. Examination of SNAP25-Htt150Q transgenic mouse brain tissues revealed that these phosphorylations were also reduced in the HD mouse brains.Many genetic mouse models of Huntington’s disease (HD) had established that mutant huntingtin accumulated in various sub cellular regions to affect a variety of cellular functions, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remaind to be determined. We generated transgenic mice that selectively express mutant huntingtin in the presynaptic terminals. Despite its low level, synaptic mutant huntingtin caused age-dependent neurological symptoms and earlier death in mice, as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fraction and immunoprecipitation of synapsin-1from HD CAG150KI mouse brain revealed mutant huntingtin bound to synapsin-1, a protein whose phosphorylation was critical for neurotransmitter release. We found that polyQ expansion enhanced the interaction of exon-1huntingtin with C-terminal region of synapsin-1to reduce synapsin-1phosphorylation. Our findings pointed to a critical role for synaptic huntingtin in the neurological symptoms of HD, providing a new therapeutic target.